1. Field of the Invention
This invention relates to the use of metallic glass-coated microwire (MGCM) in support of a polymerase chain reaction (PCR); and more particularly, to a micro-scale, self-contained PCR system that is capable of conducting the PCR (or an RT-PCR) reaction, detecting PCR products, and transmitting a signal that indicates whether or not a nucleic acid amplification event has occurred.
2. Description of the Prior Art
The genetic material of each living organism, whether plant or animal, bacterium or virus, possesses sequences of its nucleotide building blocks (usually DNA, sometimes RNA) that are uniquely and specifically present only in its own species. Indeed, complex organisms such as human beings possess DNA sequences that are uniquely and specifically present only in particular individuals. These unique variations make it possible to trace genetic material back to its origin, identifying with precision the species of the organism from which the genetic material came; and oftentimes the particular member of that species.
Such an investigation requires, however, that enough of the DNA under study is available for analysis, which is the issue that PCR addresses. PCR [R. K. Saiki, et. al., “Primer-directed Enzymatic Amplification of DNA with Thermostable DNA Polymerase”, Science 239 (1988) pp. 487-491] exploits the remarkable natural function of the enzymes known as polymerases. These enzymes are present in all living things, and their function is to copy genetic material and also to then proofread and correct the copies. PCR can characterize, analyze, and synthesize a specific piece of DNA or RNA. It works even on extremely complicated mixtures, seeking out, identifying, and duplicating a particular bit of genetic material from blood, hair, or tissue specimens, from microbes, animals, or plants, some of them many thousands, or possibly even millions of years old.
PCR requires a template molecule (the DNA or RNA to be copied) and two primer molecules to get the process started. These primers, called nucleotides or bases, are short chains of the four different chemical components that make up any strand of genetic material. DNA itself is a chain of nucleotides. Under most conditions, DNA is double-stranded, consisting of two such nucleotide chains that wind around each other in what is commonly known as a double helix. Primers are single-stranded. They consist of a string of nucleotides in a specific sequence that will, under the right conditions, bind to a specific complementary sequence of nucleotides in another piece of single-stranded RNA or DNA. For PCR, primers must be duplicates of nucleotide sequences on either side of the piece of DNA of interest, which means that the exact order of the primers' nucleotides must already be known. These flanking sequences can be constructed in a laboratory or purchased from commercial suppliers.
There are three basic steps in PCR. First, the target genetic material must be denatured; that is, the strands of its helix must be unwound and separated by heating to 90-96° C. The second step is hybridization or annealing, in which the primers bind to their complementary bases on the now single-stranded DNA. The next step is DNA synthesis by a polymerase. Starting from the primer, the polymerase can read a template strand and match it with complementary nucleotides very quickly. The result is then two new helixes in place of the first, each composed of the original strands plus its newly assembled complementary strand.
All that PCR requires in the way of equipment is a reaction tube, reagents, and a source of heat. Different temperatures have been found to be optimal for each of the three steps in the PCR reaction. Thus, commercial PCR machines have been developed to automatically control these temperature regimes automatically.
To get more of the DNA desired, the process is simply repeated by denaturing the DNA that has already been made. The amount of DNA will double with every processing cycle, each of which takes only 1-3 minutes so that repeating the process for just 45 minutes can generate hundreds of millions (usually billions) of copies of a specific DNA strand. Once the primers have been characterized and obtained, PCR can do, in one week, work that used to be technically impossible (e.g. the cloning of a single copy DNA molecule).
One of the most troublesome technical problems encountered with PCR involves contamination of the sample with extraneous genetic material, thereby generating numerous copies of irrelevant DNA. When such contamination occurs, the resulting product is rendered useless; and oftentimes leads erroneous conclusions. Preventing contamination is of particular importance in human applications, such as medicine or the law, in which someone's life may literally hang in the balance. Likewise, security and defense applications will require a very low number of ‘false positive’ events. For example, mistaken activation of a PCR-based bioweapons sensor could result in mobilization of defense forces or heighten public anxiety due to inaccurate information predicting a bioweapons-based attack.